Protein Arginine Deiminases (PADs) are calcium dependant enzymes that catalyze the hydrolytic conversion of arginine residues to citrulline residues in a variety of protein substrates as illustrated in FIG. 1. While the five known members of this family, PADs 1, 2, 3, 4, and 6, share a high level (˜50%) of sequence homology, their tissue distribution varies widely. Additionally, while, PADs 1-3 and PAD6 are primarily cytoplasmic enzymes, PAD4 is found in both cytoplasmic granules and within cell nuclei. Over the past several years, evidence has emerged suggesting that the dysregulated activity of these enzymes, most notably PAD4, plays a causative role in a number of human diseases, including rheumatoid arthritis (RA), multiple sclerosis (MS), and cancer.
Evidence linking dysregulated PAD activity to rheumatoid arthritis (RA) includes the suggestion that the PAD4 gene represents a susceptibility locus for rheumatoid arthritis (RA) in the Japanese population. While a conclusive link between PAD4 and RA in French, German, and English populations has yet to be demonstrated, the preponderance of evidence from serological and biochemical studies suggests that PAD activity, in general, plays a role in the onset and progression of RA. These data include the fact that the RA-associated HLA-DRB1*0401 MHC class II molecule binds with high affinity to a Cit-containing peptide. While the precise role of PAD4, and/or other PADs [e.g., PAD2], in the pathophysiology of RA is largely speculative, studies suggest that an elevated PAD activity is disease-causing in at least a subset of the patient population. The finding that PAD4 catalyzes the deimination of histones H2A, H3, and H4 has also drawn the attention of a broader community of scientists who are interested in characterizing the role of histone modifications in regulating gene transcription. In fact, it has recently been demonstrated that PAD4 acts as a transcriptional corepressor of the estrogen receptor and p53, and that the ability of PAD4 to alter gene transcription is peculiar to its catalytically active form.
Evidence linking dysregulated PAD activity to cancer includes the fact that PAD4 is overexpressed in variety of malignant tumors; however, this overexpression is not observed in the cells of benign tumors. Additionally, the levels of PAD4 are elevated in the blood of patients with malignant cancers, while the levels in patients with benign tumors remain normal. Interestingly, these levels decrease in patients with malignant tumors after these tumors have been resected. As in RA, the levels of citrullinated antithrombin are also elevated in patients with malignant cancers, a finding that is especially relevant given that thrombin activity increases the expression of both VEGF and integrin β3, thus contributing to angiogenesis, hyperplasia, and metastasis. PAD4 also acts as a transcriptional corepressor for p53, thus increased PAD4 activity could conceivably contribute to tumorgenesis both intra- and extracellularly.
In light of the evidence linking PAD activity to various disease states, it is conceivable, if not likely, that PAD-specific inhibitors could possess clinical utility for the treatment of RA, MS, and cancer. To date, a few inhibitors of PAD4 have been described in the literature. Of the known PAD inhibitors currently reported in the literature, two haloacetamidine-based compounds are the most potent described to date, F— and Cl— amidine (FIG. 2)—these two inactivators convalently modify the active site cysteine of the enzyme. Given the successful inhibition of PAD activity that was achieved with these inhibitors, elaboration of such structures in an effort to develop inhibitors with even greater potency would be beneficial.
In view of the above, a need exists for inhibitors with even greater potency for inhibition of PAD activity.